Recombinant Arabian camel Anti-FURIN Single Domain Antibody (PABC-557) (CAT#: PABC-557)

Figure 1 Co-immunoprecipitation of human furin with the different sdAbs RPE.

40 cells were transfected with human furin with or without expression vectors encoding the different sdAbs tagged with HA and immunoprecipitation was performed using an anti-HA antibody. Subsequently, Western blot analysis was performed using the anti-furin antibody MON152. Lanes 1, beads without anti-HA antibody; lanes 2, beads with anti-HA antibody; lanes 3, total cell lysate. Untransfected cells were included as a negative control in the last lane (-).

Dahms, S. O., Creemers, J. W., Schaub, Y., Bourenkov, G. P., Zögg, T., Brandstetter, H., & Than, M. E. (2016). The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers. Scientific reports, 6, 34303.

Figure 2 The sdAbs inhibit diphtheria-toxin-mediated cytotoxicity as efficiently as α1-PDX.

HEK-293T cells were transfected with empty vector, an expression vector encoding α1-PDX or the different sdAbs 24 h before exposure to diphtheria toxin. Then, 3 h after adding diphtheria toxin, cell viability was assessed by the MTT assay. The sdAbs protected the cells from cytotoxicity as efficiently as the well characterized furin inhibitor α1-PDX. The viability of cells treated with the diphtheria toxin together with α1-PDX or the different sdAbs is significantly higher when compared with cells only treated with the diphtheria toxin.

Dahms, S. O., Creemers, J. W., Schaub, Y., Bourenkov, G. P., Zögg, T., Brandstetter, H., & Than, M. E. (2016). The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers. Scientific reports, 6, 34303.

Figure 3 Four different sdAbs inhibit the cleavage of the furin substrates TGFβ and GPC3

The ratio of mature/precursor GPC3 was calculated from three different experiments using ImageJ software.

Zhu, J., Declercq, J., Roucourt, B., Ghassabeh, G. H., Meulemans, S., Kinne, J., ... & Muyldermans, S. (2012). Generation and characterization of non-competitive furin-inhibiting sdAbs. Biochemical Journal, 448(1), 73-82.

Figure 4 Purified sdAbs cannot inhibit the cleavage of the small pyr-Arg-Thr-Lys-Arg-AMC substrate, but do inhibit the cleavage of diphtheria toxin.

D9R and purified Nb6, Nb14 and Nb16 inhibit the furin-mediated cleavage of diphtheria toxin (DT), as shown by Coomassie Brilliant Blue staining.

Zhu, J., Declercq, J., Roucourt, B., Ghassabeh, G. H., Meulemans, S., Kinne, J., ... & Muyldermans, S. (2012). Generation and characterization of non-competitive furin-inhibiting sdAbs. Biochemical Journal, 448(1), 73-82.

Figure 5 The sdAbs inhibit diphtheria-toxin-mediated cytotoxicity as efficiently as α1-PDX and D9R.

HEK-293T cells were transfected with empty vector, an expression vector encoding α1-PDX or the different sdAbs 24 h before exposure to diphtheria toxin. Then, 3 h after adding diphtheria toxin, cell viability was assessed by the MTT assay. The sdAbs protected the cells from cytotoxicity as efficiently as the well-characterized furin inhibitor α1-PDX. The viability of cells treated with the diphtheria toxin together with α1-PDX or the different sdAbs is significantly higher when compared with cells only treated with the diphtheria toxin.

Zhu, J., Declercq, J., Roucourt, B., Ghassabeh, G. H., Meulemans, S., Kinne, J., ... & Muyldermans, S. (2012). Generation and characterization of non-competitive furin-inhibiting sdAbs. Biochemical Journal, 448(1), 73-82.

Figure 6 The sdAbs inhibit diphtheria-toxin-mediated cytotoxicity as efficiently as α1-PDX and D9R.

At 2 h prior to the exposure to diphtheria toxin, HEK-293T cells were incubated with 10 μM of purified sdAbs or with D9R. Then 1.5 h after adding the diphtheria toxin, cell viability was assessed by the MTT assay. The sdAbs targeting furin significantly protected the cells from cytotoxicity; protection was as efficient as the well-characterized furin inhibitor D9R, whereas control sdAbs which do not target furin did not inhibit cell toxicity.

Zhu, J., Declercq, J., Roucourt, B., Ghassabeh, G. H., Meulemans, S., Kinne, J., ... & Muyldermans, S. (2012). Generation and characterization of non-competitive furin-inhibiting sdAbs. Biochemical Journal, 448(1), 73-82.

Figure 7 The sdAbs inhibit diphtheria-toxin-mediated cytotoxicity as efficiently as α1-PDX and D9R.

At 30 min prior to the exposure to anthrax toxin (200 ng/ml PA and 400 ng/ml LF), RAW cells were incubated with 20 μM purified sdAbs or with 10 μM D9R. Then, 1.5 h after adding the toxins cell viablility was assayed using the MTT assay. sdAbs targeting furin significantly protected the cells from cytotoxicity although less efficient than D9R, whereas control sdAbs which do not target furin only showed a mild effect on the cell toxicity.

Zhu, J., Declercq, J., Roucourt, B., Ghassabeh, G. H., Meulemans, S., Kinne, J., ... & Muyldermans, S. (2012). Generation and characterization of non-competitive furin-inhibiting sdAbs. Biochemical Journal, 448(1), 73-82.

Figure 8 The sdAbs are non-competitive inhibitors with K i values in the micromolar range

Dixon plot for inhibition of furin-mediated cleavage of diphtheria toxin by Nb14. The reciprocal velocity is plotted against the inhibitor concentration. The trendlines drawn for each substrate concentration intersect in a single point on the x-axis, indicating non-competitive inhibition.

Zhu, J., Declercq, J., Roucourt, B., Ghassabeh, G. H., Meulemans, S., Kinne, J., ... & Muyldermans, S. (2012). Generation and characterization of non-competitive furin-inhibiting sdAbs. Biochemical Journal, 448(1), 73-82.